Prophylactic tuberculosis vaccine

ABSTRACT

The invention relates to the use of an immunotherapeutic agent containing cell wall fragments of a virulent strain of  Mycobacterium tuberculosis -complex (MTB-C) for the preparation of a drug for the prophylactic treatment of tuberculosis, in which said agent can be obtained using a method comprising the following steps: cultivate the virulent MTB-C strain over a period equal to or greater than three weeks; and, subsequently, homogenate the cell culture in the presence of a nonionic surfactant.

FIELD OF THE ART

The present invention relates to the use of an immunotherapeutic agentbased on cell wall fragments of a virulent strain of Mycobacteriumtuberculosis-complex for the preparation of a drug for the prophylactictreatment of tuberculosis.

PRIOR STATE OF THE ART

Tuberculosis is a chronic infectious disease caused by the Mycobacteriumtuberculosis-complex (MTB-C) bacilli, which currently include thespecies M. tuberculosis, M. bovis, M. microti and M. africanum.

According to the World Health Organization, 8,000,000 new cases ofpeople manifesting the disease are recorded worldwide every year andabout 3,000,000 people die. It is considered that there are more than2,000,000,000 infected people worldwide and that 90-100 million more newinfections are generated each year.

The current vaccine which is used in the preventive treatment againsttuberculosis is based on bacteria of the strain called BCG (BacillusCalmette-Guerin), an attenuated variant of M. bovis.

Various vaccines against tuberculosis based on cell wall fragments ofvirulent or avirulent strains of Mycobacterium are described in thestate of the art. It is also described that the adjuvant used in thecomposition of the vaccine greatly influences the effectiveness thereof.

E. Ribi et al., Nature 1963, 198, pages 1214 to 1215, describe theimmunization assays performed with a composition comprising cell wallfragments of the avirulent BCG strain and mineral oil. Said fragmentsare obtained by homogenisation of a culture of the mentioned strain inmineral oil. The composition is more effective than the conventionalvaccine (BCG). Nevertheless, it is described in the same article thatthe cell wall fragments do not induce any immunological response whenthey are obtained by homogenisation in water and in the absence of themineral oil.

D. P. Pal et al., Indian J. Med. Res. 1977, 65, pages 340 to 345,describe a vaccine prepared with cell wall fragments of the virulentH₃₇Rv strain and mineral oil. In this case the cell wall fragments areobtained by means of homogenisation of the dead cells in aqueous phase,and the mineral oil is subsequently added to the composition. It is alsodescribed that the cell wall fragments homogenised in aqueous phase arenot immunogenic and that the presence of mineral oil is necessary forthe vaccine to be effective.

G. K. Khuller et al., Folia Microbiol., 1992, 37, pages 407 to 412,describe the protective efficacy of different fractions of the cell wallof the avirulent H₃₇Ra strain of M. tuberculosis formulated withFreund's incomplete adjuvant, which also includes mineral oil.

E. M. Agger et al., Scand. J. Immunol., 2002, 56, pages 443 to 447,describe vaccines comprising cell wall fragments of the virulent H₃₇Rvstrain, which are effective when they include the cationic surfactantdimethyldioctadecylammonium bromide as an adjuvant. It is also describedthat the assays conducted with homogenised M. tuberculosis bacilli whichdo not contain the mentioned adjuvant do not generate levels ofresistance against tuberculosis in the murine model.

I. M. Orme Vaccine, 2006, 24, pages 2 to 19, which is a recent reviewarticle of new vaccines against tuberculosis, describes that theconventional BCG vaccine is essentially ineffective in protecting adultpeople against tuberculosis. It is indicated in the same article thatseveral candidates for different types of vaccines (subunit vaccineswith proteins, vaccines with DNA, vaccines combined with virus, vaccineswith recombinant strains) are being assayed and that new developmentsare expected.

It is therefore necessary to have a prophylactic vaccine to preventinfections caused by M. tuberculosis that is more effective than thecurrent vaccine based on the attenuated BCG strain.

OBJECT OF THE INVENTION

The object of the present invention is the use of an immunotherapeuticagent comprising cell wall fragments of a virulent strain of MTB-C forthe preparation of a drug for the prophylactic treatment of infectionscaused by M. tuberculosis.

DETAILED DESCRIPTION OF THE INVENTION

Patent application ES2231037-A1 discloses a method for the preparationof an immunotherapeutic agent comprising cell wall fragments of avirulent strain of Mycobacterium tuberculosis-complex (MTB-C). It alsodiscloses compositions containing it and the therapeutic applicationthereof for the combined treatment of tuberculosis in association withother drugs.

The authors of the present invention have discovered the use of saidimmunotherapeutic agent for the preparation of a drug for theprophylactic treatment of tuberculosis.

The object of the present invention therefore is the use of animmunotherapeutic agent comprising cell wall fragments of a virulentstrain of Mycobacterium tuberculosis-complex (MTB-C) for the preparationof a drug for the prophylactic treatment of tuberculosis, wherein saidagent is obtainable by a method comprising the following steps:

-   -   cultivate the virulent MTB-C strain over a period equal to or        greater than three weeks and, subsequently,    -   homogenate the cell culture in the presence of a nonionic        surfactant.

The virulent strain can be any virulent strain of MTB-C. One of thestrains most used by researchers in this field is called H₃₇Rv which,for example, can be freely acquired in the National Collection of TypeCultures (NCTC), London, Great Britain (deposit number NC007416).

The virulent strain can be cultivated by inoculation in culture mediawell-known by the person skilled in the art, for example Middlebrook7H10 or 7H11 agar, Sauton's medium or Proskauer-Beck medium.

The culture of the virulent strain is performed over a period equal toor greater than three weeks, preferably comprised between 3 and 4 weeks.The temperature of the culture is preferably maintained between 34° C.and 38° C.

Once the culture ends, the cells are isolated using techniques such asthose described, for example, in patent application ES2231037-A1.

The homogenisation of the live cells is carried out in the presence of anonionic surfactant, and preferably in a buffered medium at neutral pH,for example at pH comprised between 6 and 8, such as that provided byPBS buffer (phosphate buffered saline).

The homogenisation can be carried out by means of ultrasound sonication,or by means of the use of small beads of approximately 1 mm in diameter,for example, silica or zirconia/silica beads, together with a mechanicalhomogeniser. A mechanical homogeniser that can be used, for example, isthe BioSpec BeadBeater® model.

The MTB-C cells are broken by means of this homogenisation process andsmall cell wall fragments are obtained.

The type of nonionic surfactant used in the homogenisation process ispreferably selected from the group consisting of alkylphenolethoxylates, sorbitan ester ethoxylates, and mixtures thereof.

More preferably, the nonionic surfactant is selected from the group ofoctylphenol ethoxylates. More preferably, octylphenol ethoxylates withan ethylene oxide content comprised between 7 and 8 moles are used,which surfactants can be found on the market under the name TritonX-114®.

The nonionic surfactant content in the homogenisation step is preferablycomprised between 1% and 10% by weight with respect to the total weightof the homogenate, more preferably between 3% and 6% by weight.

The homogenised mass containing the cell wall fragments is subjected toa conventional treatment to separate and reject the non-fragmented cellsand the solubilized components. Centrifugation at different speeds andwashing with buffer solution as described in patent applicationES2231037-A1 can be used for example. Sediment containing the cell wallfragments is obtained after performing the mentioned purificationprocesses. Said sediment is dispersed in PBS buffer and is subjected toa conventional treatment to ensure the complete inactivation of theMTB-C cells which may have remained viable after the fragmentation andpurification process. The mentioned treatment can be a chemical process,for example by means of treatment with formaldehyde, or a physicalprocess, for example by means of autoclaving or pasteurisationtreatment.

The dispersion of cell wall fragments in PBS buffer obtained after theinactivation treatment can be lyophilised to facilitate the storagethereof. To that end, the dispersion can be distributed into vials andlyophilised at a temperature comprised between −15° C. and −25° C. andwith a vacuum comprised between 0.1 and 0.5 mbar.

The vials obtained after the lyophilisation process contain theimmunotherapeutic agent comprising the cell wall fragments of MTB-C, andthey are generally stored at very low temperatures, for example at −70°C.

As previously indicated, the object of the invention is the use of theimmunotherapeutic agent comprising cell wall fragments of a virulentstrain of MTB-C for the preparation of a drug for the prophylactictreatment of tuberculosis, i.e., for the preparation of a prophylacticvaccine against tuberculosis.

The drug for the prophylactic treatment of tuberculosis comprises theimmunotherapeutic agent based on cell wall fragments and, optionally,pharmaceutically acceptable diluents, adjuvants and/or excipients. Thedrug can be in the form of phosphate buffered saline, aqueous solution,emulsion, or in the form of liposomes.

The drug is preferably in the form of liposomes.

The liposomes can be formed using conventional auxiliary lipids andtechniques well-known by the person skilled in the art, such as thosedescribed in patent application ES2231037-A1.

The liposomes generally include phospholipids, with a neutral and/ornegative net charge, and sterols.

The phospholipids used can be, for example: phosphatidylcholine,phosphatidylserine and phosphatidylinositol.

The main component of the liposomes is usually phosphatidylcholine,which can be synthesized or isolated from natural sources. A frequentlyused commercial product is soy-derived lecithin, which is a complexmixture of phospholipids including thereamong phosphatidylcholine.

The sterols which are used in the preparation of liposomes can be, amongothers, cholesterol and bile salts.

The liposomes are preferably formed using a mixture of soy-derivedlecithin and sodium cholate.

The liposomes can optionally contain additives improving theirstability, for example: vitamin E, which acts as a lipid antioxidant.

The liposomes obtained usually have a size distribution in which 99.9%are smaller than 1 micron.

The liposomes can be subjected to lyophilisation to thus obtain theimmunotherapeutic agent in the form of lyophilised liposomes.

The drug can be administered in the form of a single dose or of severaldoses, by means of the repetition at certain time intervals. Preferablytwo doses are administered separated by a period comprised between 2 and5 weeks, preferably between 3 and 4 weeks.

The drug can be administered in a mucosa, for example, ocular,intranasal, oral, gastric, intestinal, vaginal, or urinary tract mucosa,or parenterally, for example, subcutaneously, intradermally,intramuscularly, intravenously, or intraperitoneally. Parenteraladministration is preferred.

The suitable dose depends on several parameters, including thereamongbetween the method of administration and the subject to be treated, butpreferably the dose is comprised between 1 μg and 1000 μg, morepreferably between 25 and 700, and even more preferably between 50 μgand 200 μg.

The drug comprising cell wall fragments of a virulent strain of MTB-Ccan be administered in combination with other prophylactic vaccinesagainst tuberculosis, such as those mentioned in S. H. E. Kaufmann,Nature Rev. Immunol. 2006, 6, 699-704, such as for example the BCGvaccine, subunit vaccines or recombinant BCG vaccines.

The combination of vaccines can be simultaneous or it can be done in twoinoculations separated over time. The period between the inoculationscan even be several years long.

In the case of inoculations separated over time, first a prophylacticvaccine against tuberculosis is preferably administered, and the drugcomprising the cell wall fragments of a virulent strain of MTB-C, whichacts as a re-stimulating (boost) agent of the initially inoculatedvaccine, is subsequently administered.

It has surprisingly been found that the administration of the drugcomprising the immunotherapeutic agent based on cell wall fragments of avirulent strain of MTB-C is able to induce a Th1 type interferon-γgenerating response against M. tuberculosis-specific antigens. Saidantigens include Ag85B and Ag85A, which are part of the complex Ag85,consisting of a family of low molecular weight proteins playing adecisive role in the biosynthesis of the cell wall and produced inconsiderable amounts when the bacterial cultivation is in the log phase.

It has been observed that the conventional BCG vaccine does not generatean immunoprotective response against antigens of complex Ag85, whichcould represent a lower protection capability.

It has also been found that the number of viable bacilli present in thelungs of mice vaccinated with said immunotherapeutic agent subsequentlyinfected with the virulent H₃₇Rv strain is less than the number ofviable bacilli present in the control mice group, and said number iscomparable to that of the mice vaccinated with the conventional BCGvaccine.

The examples below are shown to provide the person skilled in the artwith a detailed explanation of specific embodiments within theinvention.

Example 1 Effectiveness of the Immunotherapeutic Agent as a ProphylacticVaccine Against the Infection Caused by M. tuberculosis

The immunotherapeutic agent used in this example was prepared accordingto the method described in Example 2 of patent application ES2231037-A1.

The effectiveness of the immunotherapeutic agent based on cell wallfragments of a virulent strain of MTB-C was assayed in C57BL/6 typefemale mice from 6 to 8 weeks of age and free of specific pathogens.

The mice were divided into three groups of 12 animals each and weresubjected to the following vaccination protocol:

-   -   1) Without vaccination (control group),    -   2) Subcutaneously inoculated with two doses of 285 μg of the        immunotherapeutic agent obtained in Example 2 of patent        application ES2231037-A1 at weeks 3 and 6 of the experiment.    -   3) Subcutaneously inoculated with a dose of 2×10⁶ colony forming        units of the BCG Danish strain (Statens Serum Institute,        Denmark) at week 0 of the experiment.

The virulent strain of Mycobacterium tuberculosis (H37Rv Pasteur), whichwas cultivated in Proskauer-Beck medium until a mid-log phase, was usedfor the infection and it was stored in aliquots of 1 ml at a temperatureof −70° C. until its use.

The mice were aerosol-infected with said virulent strain at week nine ofthe experiment by means of placing them in a Middlebrook aerosolinfection apparatus which provided an inoculum of approximately 10-50viable bacilli in the lungs of the mice.

The number of viable bacilli in the lungs was determined 3 weeks afterthe aerosol infection of the animals (week 12 of the experiment)incubating serial dilutions of lung homogenate in Middlebrook 7H11 agarfor 4 weeks at 37° C. The lung was homogenized in the presence of 1 mlof twice-distilled water.

The results of Table I express the logarithm of the colony forming units(CFU) per ml which have been identified in the lung:

TABLE I Group of mice Vaccine Log₁₀CFU/ml 1 None (Control Group) 6.42 ±0.24 2 Liposome-encapsulated 5.72 ± 0.30 immunotherapeutic agent 3 BCG5.71 ± 0.58

The differences between the result of the group of mice which have notbeen vaccinated and the results of the groups vaccinated arestatistically significant.

It can be observed that in the lung of the group of mice vaccinated withthe liposome-encapsulated immunotherapeutic agent a smaller number ofviable bacilli is detected than in the lung of the mice not vaccinated,and a substantially identical number to that of the mice vaccinated withthe conventional BCG vaccine.

Therefore, the vaccination with the immunotherapeutic agent based oncell wall fragments of a virulent strain of MTB-C achieves protectingagainst infections caused by M. tuberculosis.

Example 2 Effectiveness of the Immunotherapeutic Agent as a Generator ofa Th1 Response Specific Against Infection Caused by M. tuberculosis

The immunotherapeutic agent used in this example was prepared accordingto the method described in Example 2 of patent application ES2231037-A1.The effectiveness of the immunotherapeutic agent based on cell wallfragments of a virulent strain of MTB-C was assayed in C57BL/6 typefemale mice from 6 to 8 weeks of age and free of specific pathogens inan ex vivo experiment.

The mice were divided into groups of 4 animals each and were subjectedto the following inoculation schedule:

-   -   1) Subcutaneously inoculated with saline at weeks 3 and 6 of the        experiment (control group),    -   2) Subcutaneously inoculated with two doses of 285 μg of the        immunotherapeutic agent obtained in Example 2 of patent        application ES2231037-A1 at weeks 3 and 6 of the experiment.    -   3) Subcutaneously inoculated with a dose of 2×10⁶ colony forming        units of the BCG Danish strain (Statens Serum Institute,        Denmark) at week 0 of the experiment, and with saline at weeks 3        and 6 of the experiment.    -   4) Subcutaneously inoculated with 2×10⁶ colony forming units of        the virulent strain of Mycobacterium tuberculosis (H₃₇Rv        Pasteur) at week 3 of the experiment, and with saline at week 6        of the experiment.

The mice were sacrificed at week 7, and their spleens were extracted andimmersed in tubes containing 5 ml of L-glutamine-RPMI (Gibco). The tubeswere kept in ice until the end of the experiment. The spleens weremechanically disintegrated and the suspension was filtered through anylon mesh 70 μm in diameter. Then it was centrifuged for 10 minutes at300 g. The pellets were reconstituted with 15 ml of a solutionconsisting of Tris and ammonium chloride in twice-distilled water toperform lysis on the cells. After 8 minutes they were washed with 20 mlof L-glutamine-RPMI and centrifuged for 10 minutes at 300 g.

The obtained pellets were resuspended with 5 ml of L-glutamine-RPMI, andthe suspension was filtered through a nylon mesh 70 μm in diameter. Thecells were counted with the Neubauer chamber.

The cells from the spleen of the mice were seeded on dishes at a ratioof 1×10⁵ cells/well and were cultivated with 200 μl of the completeculture medium (CCM) consisting of L-glutamine-RPMI supplemented withinactivated foetal calf serum, penicillin, streptomycin, sodiumpyruvate, and 2-mercaptoethanol, or with 200 μl of complete culturemedium (CCM) supplemented with M. tuberculosis antigens: 10 μg/ml of theantigen PPD (Statens Serum Institut, Denmark) and 5 μg/ml of ESAT-6,Ag85A and Ag85B (Lionex Diagnostics and Therapeutics GmbH, FederalRepublic of Germany).

The cells were incubated at 37° C. in an atmosphere with 5% CO₂. After96 hours, the dishes were centrifuged for 10 minutes at 300 g, and thesupernatant of each of them was collected.

The supernatants were frozen and after remaining in said state at least24 hours, the interferon-γ content was analyzed applying the doublesandwich ELISA technique and using a monoclonal antibody specific formurine interferon-γ (Diaclone).

Table II shows the average interferon-γ concentration expressed as log₁₀pg/ml which was induced in each of the groups of mice against the M.tuberculosis antigens:

TABLE II Antigens Group Inoculum Control PPD ESAT-6 Ag85B Ag85A 1Control 0 0 0 0 0 2 Liposome- 0 2.57 0 2.69⁽¹⁾ 2.53⁽¹⁾ encapsulatedimmunotherapeutic agent 3 BCG 0 2.01⁽³⁾ 0 0 0 4 H₃₇Rv 0 3.06⁽³⁾2.86⁽²⁾⁽³⁾ 2.76⁽³⁾ 2.50⁽³⁾ ⁽¹⁾Statistically significant differencesbetween Group 2 and Group 3 ⁽²⁾Statistically significant differencesbetween Group 2 and Group 4 ⁽³⁾Statistically significant differencesbetween Group 3 and Group 4

The results of Table II show that the two inoculations performed withthe liposome-encapsulated immunotherapeutic agent based on cell wallfragments of a virulent strain of M. tuberculosis are able to induce theproduction of interferon-γ against M. tuberculosis-specific antigens.This means that they induce a Th1 type protective response.

Particularly, the response induced by the group of mice inoculated withthe liposome-encapsulated immunotherapeutic agent against the antigensPPD, Ag85B and Ag85A presents no significant differences with theresponse of the group of mice inoculated with the virulent H₃₇Rv strainof M. tuberculosis. Said response is better than the response of thegroup of mice inoculated with the BCG strain, which is the conventionalvaccine, since it only induces a response against PPD and not againstAg85B and Ag85A.

Only the group of mice inoculated with the virulent strain was able toinduce a response against the antigen ESAT-6.

Therefore, the immunotherapeutic agent based on cell wall fragments of avirulent strain of M. tuberculosis is able to induce a Th1 typeprotective response against M. tuberculosis-specific antigens, which isan indicator that it can be used effectively to prevent infectionscaused by M. tuberculosis.

1.-16. (canceled)
 17. A method for the prophylactic treatment of tuberculosis, which comprises the administration of an immunotherapeutic agent comprising cell wall fragments of a virulent strain of Mycobacterium tuberculosis-complex (MTB-C), wherein said agent is obtainable by a method comprising the following steps: cultivating the virulent MTB-C strain over a period equal to or greater than three weeks and, subsequently, homogenizing the cell culture in the presence of a nonionic surfactant.
 18. The method according to claim 17, wherein the culture period is comprised between 3 and 4 weeks.
 19. The method according to claim 17, wherein the nonionic surfactant is selected from the group consisting of alkylphenol ethoxylates, sorbitan ester ethoxylates, and mixtures thereof.
 20. The method according to claim 19, wherein the nonionic surfactant is selected from the group of octylphenol ethoxylates.
 21. The method according to claim 20, wherein the nonionic surfactant is selected from the octylphenol ethoxylates with an ethylene oxide content comprised between 7 and 8 moles.
 22. The method according to claim 17, wherein the homogenisation is performed in a buffered medium at neutral pH.
 23. The method according to claim 17, wherein the method further comprises the following steps: separating the non-fragmented cells and the solubilized components by means of centrifugation, subjecting the fraction of cell wall fragments to chemical or physical treatment to inactivate the eventual virulent strain cells that it eventually contains, and drying the obtained immunotherapeutic agent by means of lyophilisation.
 24. The method according to claim 17, wherein the drug is in the form of liposomes.
 25. The method according to claim 17, wherein the drug is administered in the form of a single dose or of several doses.
 26. The method according to claim 25, wherein the drug is administered in two doses.
 27. The method according to claim 26, wherein the doses are administered separated by a period comprised between 2 and 5 weeks.
 28. The method according to claim 17, wherein the drug is administered in combination with other prophylactic vaccines against tuberculosis.
 29. The method according to claim 28, wherein the vaccines are combined in two inoculations separated over time.
 30. The method according to claim 29, wherein first a prophylactic vaccine against tuberculosis is administered, and the drug comprising the cell wall fragments of a virulent strain of MTB-C is subsequently administered. 